# Investigating 53BP1 'dephosphorylation' as a critical determinant of PARP

> **NIH NIH R01** · DANA-FARBER CANCER INST · 2020 · $385,096

## Abstract

Project Summary
53BP1 is essential for non-homologous end-joining (NHEJ) of DNA lesions and is tightly regulated by
reversible phosphorylation. We recently reported that dephosphorylation of 53BP1 at residues T1609 and
S1618 in its foci-forming region (FFR), catalyzed by the PP4C-PP4R3 phosphatase complex, is necessary for
its recruitment to double-strand breaks (DSB) during G1. Independent lines of evidence suggest that
dephosphorylation-dependent recruitment of 53BP1 to DSB could be leveraged in the context of cancer
therapy. Mutations in BRCA1, which are frequently found in breast and ovarian cancers, compromise HR and
render these tumors exquisitely sensitive to PARP inhibitors. The response to PARP inhibitors is strongly
dependent on the function of 53BP1. In fact, depletion of 53BP1 in BRCA1-mutant cells restores homologous
recombination (HR) proficiency and renders these cells resistant to PARP inhibitors. Secondly, a mutation in
53BP1 within the sequence spanningT1609/S1618 was identified in a breast cancer patient and we found that
this mutation disrupts 53BP1 recruitment to DNA damage foci, and induces resistance to PARP inhibitors.
Importantly, the functional deficiency in 53BP1 (deletion or mutation) enhances radiosensitivity. Therefore
BRCA1-mutant tumors that develop resistance to PARP inhibitors due to loss in 53BP1 function are likely to
respond to radiotherapy. Based on these results we hypothesize that the PP4C-53BP1 axis has significant
therapeutic implications specifically in BRCA1-mutant tumors. We have observed that phosphorylation of the
Ser840 residue in a fragment in the C-terminus of PP4R3 is necessary for the formation of PP4R3/53BP1
complex. In Aim 1 we will utilize phosphoproteomic methods to examine the specificity of PP4C/PP4R3
mediated 53BP1 dephosphorylation. Use in vitro binding assays and cell based assays to determine whether
the interaction of PP4R3 and 53BP1 is direct, or mediated by other factors. Finally use time-lapse imaging to
examine the kinetics of interaction of PP4R3 and 53BP1 during late mitosis and early G1. Our preliminary
results suggest that Cdk5 mediated phosphorylation of the Ser840 residue on PP4R3 is critical for the
interaction of PP4R3 and 53BP1. Aim 2 will utilize several innovative chemical genetic tools to systematically
investigate the connection of Cdk5 with the PP4/53BP1 axis. Using an extremely specific and potent Cdk5
inhibitor, we will determine the precise impact of Cdk5 on PP4R3 phosphorylation during mitosis. 53BP1 foci
formation in G1, and broadly assess its impact on DSB repair. Furthermore proteomics coupled to an unbiased
chemical genetic approach will allow us to identify other Cdk5 substrates involved in DNA repair in cells. In
Aim 3 we will identify and investigate the impact of sporadic mutations in the FFR of 53BP1, a PP4R3-S840F
mutation and inhibition of Cdk5 on olaparib sensitivity of BRCA1-mutant ovarian and breast tumor lines in vitro
and for selected on...

## Key facts

- **NIH application ID:** 9870881
- **Project number:** 5R01CA208244-04
- **Recipient organization:** DANA-FARBER CANCER INST
- **Principal Investigator:** Dipanjan Chowdhury
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $385,096
- **Award type:** 5
- **Project period:** 2017-03-15 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9870881

## Citation

> US National Institutes of Health, RePORTER application 9870881, Investigating 53BP1 'dephosphorylation' as a critical determinant of PARP (5R01CA208244-04). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9870881. Licensed CC0.

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